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  • Telomerase reactivation is a characteristic

    2018-10-24

    Telomerase reactivation is a characteristic of successful reprogramming to pluripotency and important for self-renewal in ESCs (Batista et al., 2011; Marion et al., 2009b). We attempted to suppress telomerase activity in WS iPSCs using the same BIBR 1532 concentration in WS-MSCtert. However, telomerase is expressed so abundantly in iPSCs that less than 20% of inhibition was achieved. At this level no change in morphology, decrease of SSEA4 (a surface marker for iPSCs), or BrdU incorporation was observed (Figure S1G). Upon differentiation and lineage specification, telomerase is “programmed” to different activity levels, and thus different cells show variable vulnerability to aging. Differential telomerase activity in various stem cells has been documented (Dolci et al., 2002; Morrison et al., 1996; Zimmermann et al., 2003). We compared three different stem cell types: the hESCs derived from the embryo, the primary MSCs derived from adult bone marrow, and the NPCs derived from the cortical region of human fetal VE-822 for their telomerase activity. The result confirmed a notable difference in telomerase activity among these stem cell types (Figure S2C). In addition to telomerase, the shelterin complex caps and stabilizes the telomeres (Palm and de Lange, 2008). Interestingly, a comparison of shelterin gene expression in the derived normal/WS stem cells indicated a significant difference for some of the capping genes, such as TRF1, TRF2, and POT1 (Figure S2B). Coincidently, WRN cooperates with POT1, TRF1, and TRF2 for proper telomere maintenance (Opresko et al., 2002, 2004, 2005). Among them, TRF2 and POT1 are known to repress ATM/ATR cascades in response to uncapping of chromosomal ends (Denchi and de Lange, 2007). TRF1 is abundantly expressed in pluripotent stem cells and restricted to some other adult stem cells, as TRF1 is a direct transcriptional target of Oct3/4 (Schneider et al., 2013). These observations suggest that by modulating the telomere-regulating machinery, it is possible to rescue or slow the accelerated aging. Next, targeting the tumor suppressor p53 can mechanically rescue senescence; however, it increases the incidence of tumorigenesis and genomic instability. A major cause in patients with WS is the development of mesenchymal cancers, such as soft tissue sarcoma and osteosarcoma (Chen and Oshima, 2002). It is proposed that immortalization of mesenchymal cancers in WS is acquired by alternative lengthening of the telomere (ALT) instead of telomerase activation (Laud et al., 2005; Multani and Chang, 2007). Although we did not observe immortalization of MSC cultures, our cell model may provide an opportunity to study the unique feature of tumorigenesis in WS. In the present study we focused on stem or progenitors cells; the exhaustion of these progenitors is believed to arise with organismal aging. Terminally differentiated cells, such as fibroblasts, bone cells, and endothelial and smooth muscle cells, as contrasted to neurons, may provide more insight into the underlying mechanism of accelerated aging.
    Experimental Procedures
    Acknowledgments
    Introduction To ensure that all lineages will develop after fertilization, germ cells must proceed through gametogenesis while maintaining totipotency and resisting somatic differentiation. After their induction, mammalian primordial germ cells (PGCs) express the transcription factors sufficient to not only maintain their pluripotency, such as Oct4, Sox2, or Nanog, but also activate the epigenetic changes essential to PGC specification, including chromosome X inactivation, histone H3K9 demethylation, and genome-wide erasure of methylated DNA (reviewed in Magnúsdóttir et al., 2012). The use of nonvertebrate systems such as C. elegans or D. melanogaster to study germ cell specification revealed that combinations of genetic and epigenetic events were the key to somatic fate repression. To maintain their unique status, C. elegans PGCs globally repress mRNA transcription and establish a specific chromatin structure and composition to tightly control gene expression (Wang and Seydoux, 2013). Recently, germline reprogramming was “artificially” obtained by the simultaneous ectopic expression of master somatic fate inducers (“terminal selector genes”) and the downregulation of chromatin repressors such as LIN-53/RbAP46-48 and the H3K27 methyl-transferase Polycomb (Patel et al., 2012; Tursun et al., 2011), implying that specific combinations of transcriptional and epigenetic factors were capable of controlling the germ cell program.